Laboratory of Aquatic Biomedicine

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Work in this laboratory centers on comparative immunopathology and molecular biology using marine invertebrates as experimental models. Examples of current research include determining the prevalence of leukemia in Mya arenaria (soft shell clam) in Massachusetts. Monoclonal antibodies to molluscan tumor cells and invertebrate viruses have been developed to detect disease-associated antigens at the cell surface. Immunochemistry and protein chemistry focus on determining whether cell surface antigens are preserved during phylogeny and if they serve unique functions in self, non-self recognition. Work in molecular biology is creating a clearer understanding of the comparative etiology and pathogenesis of tumors, particularly in environmentally impacted aquatic animals.


Recent Submissions

Now showing 1 - 5 of 5
  • Article
    The nerve hemoglobin of the bivalve mollusc Spisula solidissima : molecular cloning, ligand binding studies, and phylogenetic analysis
    (American Society for Biochemistry and Molecular Biology, 2005-12-13) Dewilde, Sylvia ; Ebner, Bettina ; Vinck, Evi ; Gilany, Kambiz ; Hankeln, Thomas ; Burmester, Thorsten ; Kreiling, Jill A. ; Reinisch, Carol L. ; Vanfleteren, Jacques R. ; Kiger, Laurent ; Marden, Michael C. ; Hundahl, Christian ; Fago, Angela ; Van Doorslaer, Sabine ; Moens, Luc
    Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (µM). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mM), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analogy, the histidine E7 is proposed as a sixth ligand. Kinetic and equilibrium measurements show a moderate oxygen affinity (P50 ~0.6 torr) and no cooperativity. The histidine binding affinity is 100-fold lower than in neuroglobin. Phylogenetic analysis demonstrates a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. We conclude that invertebrate nerve Hbs expressed at high levels are, despite the hexacoordinate nature of their heme iron, not essentially different from other intracellular Hbs. They most likely fulfill a myoglobin-like function and enhance oxygen supply to the neurons
  • Preprint
    Identification of DeltaN isoform and polyadenylation site choice variants in molluscan p63/p73 -like homologues
    ( 2006-07-25) Muttray, Annette F. ; Cox, Rachel L. ; Reinisch, Carol L. ; Baldwin, Susan A.
    The p53 family of transcription factors has been implicated in many vertebrate cancers. Altered p53 and p73 protein expression observed in leukemic cells of mollusks suggests that these transcription factors might be involved in invertebrate cancers as well. Here, we fully characterize the mRNA of four novel p53-like variants in the bivalve mollusks Mytilus trossulus (bay mussel) and Mytilus edulis (blue mussel). These species, widely used for environmental assessment, develop a haemic neoplasia (leukemia) that is frequently fatal. The correlation between expression of p53 and its close relative p73 and onset of molluskan leukemia was documented previously. We report the sequences of two distinct and novel p63/p73-like mRNAs, amplified by polymerase chain reaction (PCR) from both species. One of the p63/p73-like isoforms contains a 360 nt truncation in the 5' coding region. Based on this truncation and concomitant lack of a trans-activation (TA) domain, we designate this variant as a DeltaNp63/p73-like isoform: the first to be reported in an invertebrate species. In mammalian species, DeltaNp73 potently inhibits the tumor-suppressive function of p73 and p53, and its over-expression serves as a robust marker for mammalian cancer. In addition, we report on the occurrence of alternate polyadenylation sites in the molluskan p63/p73: one proximal and one distal site, which differ by 1260 nt. We hypothesize that differential expression of various molluskan p63/p73-like isoforms, controlled in part by polyadenylation site choice variation, may help to interpret the apparently opposing roles of this gene in the development of cancer. Overall, this research further illustrates the utility of the molluskan model for studies involving the molecular mechanisms of oncogenesis in naturally occurring populations. The data presented here require a revisiting of hypotheses regarding evolution of the p53 gene family. Current hypotheses indicate that 1) the protostome gene family does not contain an intronic promoter for DeltaN expression and 2) p53 gene duplication did not occur in protostomes. Our characterization of DeltaN p63/73 in mussel suggests that molluskan p53 gene family members have acquired an intronic promoter or splicing mechanism, either by invention that predates the evolutionary split of deuterostoms from protostomes, or by parallel evolution. Our data also show that Mytilus p53, p63/p73 and DeltaNp63/p73 are identical in their core regions with variation limited to their C- and N-terminals. This supports the notion that alternative splicing, intronic promoter usage and polyadenylation site choice may lead to expression of distinct isoforms originating from one common gene.
  • Preprint
    Identification and phylogenetic comparison of p53 in two distinct mussel species (Mytilus)
    ( 2005-02-10) Muttray, Annette F. ; Cox, Rachel L. ; St-Jean, Sylvie D. ; van Poppelen, Paul ; Reinisch, Carol L. ; Baldwin, Susan A.
    The extent to which humans and wildlife are exposed to anthropogenic challenges is an important focus of environmental research. Potential use of p53 gene family marker(s) for aquatic environmental effects monitoring is the long-term goal of this research. The p53 gene is a tumor suppressor gene that is fundamental in cell cycle control and apoptosis. It is mutated or differentially expressed in about 50% of all human cancers and p53 family members are differentially expressed in leukemic clams. Here, we report the identification and characterization of the p53 gene in two species of Mytilus, Mytilus edulis and Mytilus trossulus, using RT-PCR with degenerate and specific primers to conserved regions of the gene. The Mytilus p53 proteins are 99.8% identical and closely related to clam (Mya) p53. In particular, the 3′ untranslated regions were examined to gain understanding of potential post-transcriptional regulatory pathways of p53 expression. We found nuclear and cytoplasmic polyadenylation elements, adenylate/uridylate-rich elements, and a K-box motif previously identified in other, unrelated genes. We also identified a new motif in the p53 3′UTR which is highly conserved across vertebrate and invertebrate species. Differences between the p53 genes of the two Mytilus species may be part of genetic determinants underlying variation in leukemia prevalence and/or development, but this requires further investigation. In conclusion, the conserved regions in these p53 paralogues may represent potential control points in gene expression. This information provides a critical first step in the evaluation of p53 expression as a potential marker for environmental assessment.
  • Article
    Detecting p53 family proteins in haemocytic leukemia cells of Mytilus edulis from Pictou Harbour, Nova Scotia, Canada
    (National Research Council Canada, 2005-09-01) St-Jean, Sylvie D. ; Stephens, Raymond E. ; Courtenay, S. C. ; Reinisch, Carol L.
    Evaluating patterns of expression of p53-related proteins in cells is a novel approach in defining environmentally linked diseases. We have examined the induction of haemocytic leukemia in Mytilus edulis by municipal and industrial contaminants in Pictou Harbour, Nova Scotia, Canada. We used a murine monoclonal antibody, 1E10, as a diagnostic reagent to detect leukemic cells. We first characterized the reactivity of 1E10 with both normal and leukemic Mytilus haemocytes by confocal microscopy. We then compared p53 gene family expression (p53, p63–p73, and p97) in normal versus leukemic haemocytes using a panel of monoclonal and polyclonal antibodies to p53 family proteins. The immunochemical data demonstrate that haemocytic leukemia cells of M. edulis differentially express p63–p73 and p97–p120 proteins. We subsequently used 1E10 to diagnose haemocytic leukemia in 500 M. edulis previously deployed 6 months earlier in Pictou Harbour. In the field, Mytilus caged near untreated municipal wastewater and bleached kraft pulpmill effluents have a significantly greater chance of developing haemocytic leukemia than do mussels exposed to reference sites.
  • Article
    A New invertebrate member of the p53 gene family is developmentally expressed and responds to polychlorinated biphenyls
    (National Institute of Environmental Health Sciences, 2002-03-07) Jessen-Eller, Kathryn ; Kreiling, Jill A. ; Begley, Gail S. ; Steele, Marjorie E. ; Walker, Charles W. ; Stephens, Raymond E. ; Reinisch, Carol L.
    The cell-cycle checkpoint protein p53 both directs terminal differentiation and protects embryos from DNA damage. To study invertebrate p53 during early development, we identified three differentially expressed p53 family members (p53, p97, p120) in the surf clam, Spisula solidissima. In these mollusks, p53 and p97 occur in both embryonic and adult tissue, whereas p120 is exclusively embryonic. We sequenced, cloned, and characterized p120 cDNA. The predicted protein, p120, resembles p53 across all evolutionarily conserved regions and contains a C-terminal extension with a sterile alpha motif (SAM) as in p63 and p73. These vertebrate forms of p53 are required for normal inflammatory, epithelial, and neuronal development. Unlike clam p53 and p97, p120 mRNA and protein levels are temporally expressed in embryos, with mRNA levels decreasing with increasing p120 protein (R2 = 0.97). Highest surf clam p120 mRNA levels coincide with the onset of neuronal growth. In earlier work we have shown that neuronal development is altered by exposure to polychlorinated biphenyls (PCBs), a neurotoxic environmental contaminant. In this study we show that PCBs differentially affect expression of the three surf clam p53 family members. p120 mRNA and protein are reduced the most and earliest in development, p97 protein shows a smaller and later reduction, and p53 protein levels do not change. For the first time we report that unlike p53 and p97, p120 is specifically embryonic and expressed in a time-dependent manner. Furthermore, p120 responds to PCBs by 48 hr when PCB-induced suppression of the serotonergic nervous system occurs.